Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase.

Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson's disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a "floor plate protocol" generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH+ cells (≈30%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH+ neurons compared to the TH- cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH+ and TH- neuronal cell populations and that the "floor plate protocol" is particularly efficient in generating electrophysiologically mature TH+ DNs, which are the most vulnerable neuronal subtype in PD.

Isolation and transfection of myenteric neurons from mice for patch-clamp applications.

The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent electrophysiological characterization of individual neurons in current-clamp and voltage-clamp recordings. With this protocol, ENS neurons can be prepared, transfected, and electrophysiologically characterized within 72 h. Using isolated ENS neurons, we demonstrate the feasibility of the approach by functional overexpression of recombinant voltage-gated NaV1.9 mutant channels associated with hereditary sensory and autonomic neuropathy type 7 (HSAN-7), a disorder characterized by congenital analgesia and severe constipation that can require parenteral nutrition. Although our focus is on the electrophysiological evaluation of isolated ENS neurons, the presented methodology is also useful to analyze molecules other than sodium channels or to apply alternative downstream assays including calcium imaging, proteomic and nucleic acid approaches, or immunochemistry.

Hypoxia-induced mitogenic factor has antiapoptotic action and is upregulated in the developing lung: coexpression with hypoxia-inducible factor-2alpha.

Hypoxia-induced mitogenic factor (HIMF), also called FIZZ1 or RELMalpha, was a newly found cytokine. Hypoxia caused robust HIMF induction in the lung, and HIMF has potent pulmonary vasoconstrictive, proliferative, and angiogenic properties. To investigate the role of HIMF in lung development, we determined its spatial and temporal expression. From embryonic day (E)16 to postnatal day (P)28, HIMF was strongly expressed in the cytoplasm of bronchial epithelial cells, type II cells, endothelial cells, and primitive mesenchymal cells. Treatment with HIMF resulted in a significant reduction of apoptosis in cultured embryonic lung, thus revealing a previously unknown function of HIMF. Because HIMF gene is upregulated by hypoxia and contains a hypoxia-inducible transcription factor (HIF) binding site, we subsequently investigated whether HIMF was coexpressed with HIF-2alpha or HIF-1alpha. HIF-1alpha expression was temporally distinct from HIMF expression. In contrast, HIF-2alpha was present in endothelial cells, bronchial epithelial cells, and type II cells from E18 to P28. Thus, HIMF and HIF-2alpha were temporally and spatially coexpressed in the developing lung. These results indicate a role for HIMF in lung development, possibly under the control of HIF-2, and suggest that HIMF regulates apoptosis and may participate in lung alveolarization and maturation.